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Purpose: The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor–resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies.Experimental Design: We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways.Results: Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor–resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents.Conclusions: This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.

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Supplementary Figures S1-S2 from Vascular Endothelial Growth Factor Receptor-1 Contributes to Resistance to Anti–Epidermal Growth Factor Receptor Drugs in Human Cancer Cells

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Purpose:K-Ras somatic mutations are a strong predictive biomarker for resistance to epidermal growth factor receptor (EGFR) inhibitors in patients with colorectal and pancreatic cancer. We previously showed that the novel Toll-like receptor 9 (TLR9) agonist immunomodulatory oligonucleotide (IMO) has a strong in vivo activity in colorectal cancer models by interfering with EGFR-related signaling and synergizing with the anti-EGFR monoclonal antibody cetuximab.Experimental Design: In the present study, we investigated, both in vitro and in vivo, the antitumor effect of IMO alone or in combination with cetuximab in subcutaneous colon and orthotopic pancreatic cancer models harboring K-Ras mutations and resistance to EGFR inhibitors.Results: We showed that IMO was able to significantly restore the sensitivity of K-Ras mutant cancer cells to cetuximab, producing a marked inhibition of cell survival and a complete suppression of mitogen—activated protein kinase phosphorylation, when used in combination with cetuximab. IMO interfered with EGFR-dependent signaling, modulating the functional interaction between TLR9 and EGFR. In vivo, IMO plus cetuximab combination caused a potent and long-lasting cooperative antitumor activity in LS174T colorectal cancer and in orthotopic AsPC1 pancreatic cancer. The capability of IMO to restore cetuximab sensitivity was further confirmed by using K-Ras mutant colorectal cancer cell models obtained through homologous recombination technology.Conclusions: We showed that IMO markedly inhibits growth of K-Ras mutant colon and pancreatic cancers in vitro and in nude mice and cooperates with cetuximab via multiple mechanisms of action. Therefore, we propose IMO plus cetuximab as a therapeutic strategy for K-Ras wild-type as well for K-Ras mutant, cetuximab-resistant colorectal and pancreatic cancers. Clin Cancer Res; 17(20); 6531–41. ©2011 AACR.

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Purpose: Resistance to anti-HER2 monoclonal antibody trastuzumab is a relevant issue in breast cancer patients. Among the mechanisms implicated in trastuzumab resistance, increasing evidence supports a role of tumor microenvironment. We previously found that a novel toll-like receptor 9 agonist, referred to as immune modulatory oligonucleotide (IMO) and currently under clinical investigation, acts through epidermal growth factor receptor (EGFR) and shows direct antiangiogenic effects by cooperating with anti-EGFR or anti-VEGF drugs, thus interfering with cancer cells and microenvironment.Experimental Design: In this study, we used KPL-4 and JIMT-1 trastuzumab-resistant breast cancer cells to evaluate the combination IMO plus trastuzumab as a therapeutic option for trastuzumab-resistant breast cancers.Results: IMO inhibits KPL-4 and JIMT-1 xenografts growth and potentiates trastuzumab antitumor effect, with complete suppression of tumor growth, potent enhancement of trastuzumab-mediated antibody-dependent cell-mediated cytotoxicity, and strong inhibition of EGFR/HER2-related signaling. In KPL-4 xenografts, IMO alone interferes with HER signal transduction, whereas trastuzumab is ineffective. IMO induces an HER-dependent signal inhibition also in vitro by modulating a functional interaction between toll-like receptor 9 and HER receptors occurring at membrane level. Finally, IMO plus trastuzumab produces a cooperative antiangiogenic effect related to suppression of endothelial HER-related signaling.Conclusions: We showed a cooperative effect of IMO plus trastuzumab in trastuzumab-resistant breast cancers due to IMO direct antitumor and antiangiogenic activity and antibody-dependent cell-mediated cytotoxicity enhancement. Moreover, we provided first evidence of a toll-like receptor 9/HER interaction at membrane level as novel mechanism of action. Altogether, we propose IMO plus trastuzumab as an effective strategy in trastuzumab-resistant breast cancers. (Clin Cancer Res 2009;15(22):692130)

Purpose: The resistance to selective EGFR inhibitors involves the activation of alternative signaling pathways, and Akt activation and VEGF induction have been described in EGFR inhibitor–resistant tumors. Combined inhibition of EGFR and other signaling proteins has become a successful therapeutic approach, stimulating the search for further determinants of resistance as basis for novel therapeutic strategies. Experimental Design: We established human cancer cell lines with various degrees of EGFR expression and sensitivity to EGFR inhibitors and analyzed signal transducers under the control of EGFR-dependent and EGFR-independent pathways. Results: Multitargeted inhibitor vandetanib (ZD6474) inhibited the growth and the phosphorylation of Akt and its effector p70S6 kinase in both wild-type and EGFR inhibitor–resistant human colon, prostate, and breast cancer cells. We found that the resistant cell lines exhibit, as common feature, VEGFR-1/Flt-1 overexpression, increased secretion of VEGF and placental growth factor, and augmented migration capabilities and that vandetanib is able to antagonize them. Accordingly, a new kinase assay revealed that in addition to VEGF receptor (VEGFR)-2, RET, and EGFR, vandetanib efficiently inhibits also VEGFR-1. The contribution of VEGFR-1 to the resistant phenotype was further supported by the demonstration that VEGFR-1 silencing in resistant cells restored sensitivity to anti-EGFR drugs and impaired migration capabilities, whereas exogenous VEGFR-1 overexpression in wild-type cells conferred resistance to these agents. Conclusions: This study shows that VEGFR-1 contributes to anti-EGFR drug resistance in different human cancer cells. Moreover, vandetanib inhibits VEGFR-1 activation, cell proliferation, and migration, suggesting its potential utility in patients resistant to EGFR inhibitors.

Inhibition of a single transduction pathway is often inefficient due to activation of alternative signalling. The mammalian target of rapamycin (mTOR) is a key intracellular kinase integrating proliferation, survival and angiogenic pathways and has been implicated in the resistance to EGFR inhibitors. Thus, mTOR blockade is pursued to interfere at multiple levels with tumour growth. We used everolimus (RAD001) to inhibit mTOR, alone or in combination with anti-EGFR drugs gefitinib or cetuximab, on human cancer cell lines sensitive and resistant to EGFR inhibitors, both in vitro and in vivo. We demonstrated that everolimus is active against EGFR-resistant cancer cell lines and partially restores the ability of EGFR inhibitors to inhibit growth and survival. Everolimus reduces the expression of EGFR-related signalling effectors and VEGF production, inhibiting proliferation and capillary tube formation of endothelial cells, both alone and in combination with gefitinib. Finally, combination of everolimus and gefitinib inhibits growth of GEO and GEO-GR (gefitinib resistant) colon cancer xenografts, activation of signalling proteins and VEGF secretion. Targeting mTOR pathway with everolimus overcomes resistance to EGFR inhibitors and produces a cooperative effect with EGFR inhibitors, providing a valid therapeutic strategy to be tested in a clinical setting.

Synthetic agonists of Toll-like receptor 9 (TLR9), a class of agents that induce specific immune response, exhibit antitumor activity and are currently being investigated in cancer patients. Intriguingly, their mechanisms of action on tumor growth and angiogenesis are still incompletely understood. We recently discovered that a synthetic agonist of TLR9, immune modulatory oligonucleotide (IMO), acts by impairing epidermal growth factor receptor (EGFR) signaling and potently synergizes with anti-EGFR antibody cetuximab in GEO human colon cancer xenografts, whereas it is ineffective in VEGF-overexpressing cetuximab-resistant GEO cetuximab-resistant (GEO-CR) tumors. VEGF is activated by EGFR, and its overexpression causes resistance to EGFR inhibitors. Therefore, we used IMO and the anti-VEGF antibody bevacizumab as tools to study IMO's role on EGFR and angiogenesis and to explore its therapeutic potential in GEO, LS174T, and GEO-CR cancer xenografts. We found that IMO enhances the antibody-dependent cell-mediated cytotoxicity (ADCC) activity of cetuximab, that bevacizumab has no ADCC, and IMO is unable to enhance it. Nevertheless, the IMO-plus-bevacizumab combination synergistically inhibits the growth of GEO and LS174T as well as of GEO-CR tumors, preceded by inhibition of signaling protein expression, microvessel formation, and human, but not murine, VEGF secretion. Moreover, IMO inhibited the growth, adhesion, migration, and capillary formation of VEGF-stimulated endothelial cells. The antitumor activity was irrespective of the TLR9 expression on tumor cells. These studies demonstrate that synthetic agonists of TLR9 interfere with growth and angiogenesis also by EGFR- and ADCC-independent mechanisms affecting endothelial cell functions and provide a strong rationale to combine IMO with bevacizumab and EGFR inhibitory drugs in colon cancer patients.

Purpose: K-Ras somatic mutations are a strong predictive biomarker for resistance to epidermal growth factor receptor (EGFR) inhibitors in patients with colorectal and pancreatic cancer. We previously showed that the novel Toll-like receptor 9 (TLR9) agonist immunomodulatory oligonucleotide (IMO) has a strong in vivo activity in colorectal cancer models by interfering with EGFR-related signaling and synergizing with the anti-EGFR monoclonal antibody cetuximab. Experimental Design: In the present study, we investigated, both in vitro and in vivo, the antitumor effect of IMO alone or in combination with cetuximab in subcutaneous colon and orthotopic pancreatic cancer models harboring K-Ras mutations and resistance to EGFR inhibitors. Results: We showed that IMO was able to significantly restore the sensitivity of K-Ras mutant cancer cells to cetuximab, producing a marked inhibition of cell survival and a complete suppression of mitogen—activated protein kinase phosphorylation, when used in combination with cetuximab. IMO interfered with EGFR-dependent signaling, modulating the functional interaction between TLR9 and EGFR. In vivo, IMO plus cetuximab combination caused a potent and long-lasting cooperative antitumor activity in LS174T colorectal cancer and in orthotopic AsPC1 pancreatic cancer. The capability of IMO to restore cetuximab sensitivity was further confirmed by using K-Ras mutant colorectal cancer cell models obtained through homologous recombination technology. Conclusions: We showed that IMO markedly inhibits growth of K-Ras mutant colon and pancreatic cancers in vitro and in nude mice and cooperates with cetuximab via multiple mechanisms of action. Therefore, we propose IMO plus cetuximab as a therapeutic strategy for K-Ras wild-type as well for K-Ras mutant, cetuximab-resistant colorectal and pancreatic cancers. Clin Cancer Res; 17(20); 6531–41. ©2011 AACR.

“Targeted therapy” designates a new generation of antitumor agents designed to interfere with a specific molecular target believed to have a critical role in tumor growth or progression. Most of these targets are represented by kinases controlling cell homeostasis, such as tyrosine kinase receptors (TKRs). The epidermal growth factor receptor (EGFR) is one of the most investigated TKRs frequently expressed in a variety of epithelial tumors and correlates with poor prognosis. Several efforts have been made in the last 20 years to design therapeutic agents that inhibit EGFR, such as monoclonal antibodies (MAbs) or tyrosine kinase inhibitors (TKIs). Cetuximab is the first anti-EGFR monoclonal antibody approved by the FDA for the treatment of patients with EGFR-expressing, metastatic colorectal carcinoma, and head and neck cancer. Other MAbs directed against EGFR, including panitumumab, are in clinical development for the treatment of various human cancer types.

Background: The ‘targeted therapy’ has been defined as an innovative therapy designed to interfere with molecular targets playing a critical role in tumour growth or progression. The epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor mediating the effect of growth factors both in physiological and pathological conditions; aberrations in the EGFR may result in the development of a tumour phenotype. Objective: To summarise the current state of the development of anti-EGFR drugs. Methods: Patent literature and preclinical/clinical studies about EGFR inhibitors are analysed. Conclusion: Some monoclonal antibodies and small-molecule tyrosine kinase inhibitors have been approved in several countries for the treatment of various human cancer types; moreover, more than ten EGFR-targeting agents are actually in advanced clinical development.

Background. Somatic mutations in the K-Ras gene, leading to constitutive activation of the Ras/MAPK signalling cascade, represent a strong predictive biomarker for resistance to Epidermal Growth Factor Receptor (EGFR) inhibitors in colorectal and pancreatic cancer patients. We previously demonstrated that a novel Toll-like Receptor 9 (TLR9) agonist currently under clinical development, IMO, has a strong in vivo activity in colorectal cancer models interfering with EGFR-related signalling, synergizing with the anti-EGFR monoclonal antibody cetuximab and boosting its ADCC activity. Methods. In this study, we investigated IMO antitumor effect in colon and pancreatic cancer models with resistance to EGFR inhibitors due to K-Ras mutations. We evaluated the in vitro activity of IMO, alone or in combination with cetuximab, on growth, survival and EGFR-dependent signal transduction of cetuximab-sensitive and cetuximab-resistant colon and pancreatic cancer cells. We also investigated the antitumor effect of the combination IMO plus cetuximab on in vivo growth of orthotopically injected pancreatic cancer models. Results. We verified that K-Ras mutant colon and pancreatic cancer cell lines exhibit lower sensitivity to the anti-EGFR drugs cetuximab and erlotinib compared to K-Ras wild-type cell lines. IMO moderately inhibits in vitro growth of colon and pancreatic cancer cells, regardless of both TLR9 expression levels and K-Ras status. Surprisingly, IMO is able to partially restore cetuximab sensitivity of K-Ras mutant cancer cells. In fact, a strong inhibition of cell survival and a total suppression of MAPK phosphorylation/activation was observed with the combination IMO plus cetuximab in resistant cancer cells. IMO capability to interfere with EGFR-dependent signalling in K-Ras mutant, cetuximab-resistant cells seems to be related to modulation of a functional interaction between TLR9 and EGFR occurring at membrane level. The antitumor effect of the combination IMO plus cetuximab was confirmed also in vivo. In the luciferase- tagged AsPC1 (K-Rasmut) pancreatic cancer orthotopic model, IMO plus cetuximab causes a strong reduction of tumor burden and a significant increase in mice survival as compared to single agent treatments. Conclusion. We demonstrated for the first time that the TLR9 agonist IMO is effective in K-Ras mutant colon and pancreatic cancers, strongly inhibiting tumor growth and cooperating with cetuximab. This effect is due to multiple mechanisms of action, including interference with EGFR signalling by modulation of a TLR9/EGFR interaction and enhancement of cetuximab ADCC. Therefore, we suggest that IMO plus cetuximab may be a potentially effective therapeutic strategy for K-Ras mutant, cetuximab-resistant colorectal and pancreatic cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 615.